This study was carried out to evaluate spontaneous renal regeneration after stopping colistin methanesulfonate (CMS), which induces tubular damage, and the curative effect of Vitamin E (vit E) in rats. Animals were given the following: sterile saline (n = 6), 300,000 IU/kg/ day of CMS (n = 24), or 450,000 IU/kg/day of CMS (n = 24) for seven days. Each CMS group was subdivided into four subgroups (n = 6) and sacrificed as follows: (i) 12 h after stopping CMS, (ii) two weeks after stopping CMS, (iii) two weeks after stopping treatment with vit E, and (iv) two weeks after stopping treatment with olive oil. Subsequently, plasma creatinine (pCr), urine N-acetyl-b-D-glucosaminidase (NAG), renal tissue level of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione reductase (GSH), and renal histology were tested. CMS-induced tubular damage increased the NAG and MDA levels and decreased the SOD and GSH activities. After two weeks of stopping CMS, there was no significant renal recovery. However, treatment with vit E improved tubular regeneration and reduced the biochemical impairments. Two weeks might not be long enough for significant spontaneous renal regeneration. Improvement of renal parameters by vit E could be explained by the reduction of oxidative stress damage.

Colistin is an old class of polypeptide cationic antibiotic which is widely used after the appearance of Gram-negative bacteria, resistant to almost classes of commercially available antibiotics.[1],[2] Nephrotoxicity is the most frequently observed side effect and results in either early discontinuation of treatment or worse prognosis.[1],[2],[3] The mechanism of colistin nephrotoxicity still remains unknown; nonetheless, it has been reported to be related to the total dose of colistin and duration of therapy.[4] Tubular damage due to colistin has been suggested to be reversible on cessation of therapy.[5],[6] Nevertheless, renal reversibility after stopping colistin has not been investigated yet in an experimental setting. In patients, it is rarely mentioned due to normalization of plasma creatinine (pCr).[7],[8]

Antioxidants are well known for offering therapeutic opportunities.[9],[10],[11] Vitamin E (Vit E), a lipid-soluble compound, is a well-known antioxidant. Several reports have indicated the promising effect of vit E on drug-induced nephrotoxicity such as gentamicin, vancomycin, and cisplatin.[12],[13],[14]

Therefore, we aimed in the present study on rats, to investigate the spontaneous renal reversibility after stopping colistin and the effect of the treatment with vit E.

Material and Methods

Chemical products

Clinically, colistin is administered parenterally as sodium colistin methanesulfonate (CMS), an inactive prodrug that is converted into colistin, the antibacterial, and toxic entity.[15],[16] CMS was obtained from Aventis-France (1 million IU/vial). Interestingly enough, 1 mg of CMS is equivalent to 0.41 mg of colistin or 12,500 IU of CMS.

Male Wistar rats weighing 250 ± 20 g were purchased from the breeding center of the Central Pharmacy (SIPHAT). All animal procedures were conducted in strict conformity with the local Institute Ethical Committee Guidelines for the Care and Use of laboratory animals of our institution: they were kept in an environmentally controlled breeding room (temperature: 22°C ± 2°C, humidity: 60% ± 5%, 12 h dark/light cycle). All rats had free access to tap water and food.

Experimental design

In our previous studies on rats,[17],[18] the treatment with 300,000 IU/kg/day and 450,000 IU/kg/day of CMS led to tubular damage. This study aimed to examine the spontaneous renal recovery after stopping CMS and the effect of treatment with vit E.

Animals were randomly divided into nine groups (n = 6) as follows:

G1: were given 1 mL/kg/day of sterile saline for seven days and were sacrificed after 12 h

G2 and G3: received 300,000 IU/kg/day and 450,000 IU/kg/day of CMS for seven days, respectively, and were sacrificed after 12 h

G4 and G5: received 300,000 IU/kg/day and 450,000 IU/kg/day of CMS for seven days, respectively, and were sacrificed after two weeks;

G6 and G7: received 300,000 IU/kg/day and 450,000 IU/kg/day of CMS for seven days, respectively, were treated with vit E for two weeks and sacrificed after 12h

G8 and G9: received 300,000 IU/kg/day and 450,000 IU/kg/day of CMS for seven days, respectively, were treated with olive oil (OO) for two weeks and sacrificed after 12 h.

Sterile saline and CMS were injected intramuscularly in twice daily doses (12 h apart). Vit E was dissolved in 1 mL/kg of OO and injected subcutaneously in once daily dose. The dose of vit E used (100 mg/kg/day) had been reported earlier to be effective against nephrotoxicity induced by vancomycin.[13]

Preparation of urine, blood and renal tissues samples

At the end of each experiment period, animals were housed in individual metabolic cages, and 12 h urine samples were collected and centrifuged at 1000 g for 5 min.[17],[19] The supernatant was aliquoted into eppendorf tubes for determination of NAG level.

Thereafter, animals were anesthetized, sacrificed, and blood samples were collected from the heart in heparin tubes and centrifuged at 2500 g for 15 min.[18],[20] The plasma was aliquoted into eppendorf tubes for determination of Cr level.

The kidneys were then removed, 500 mg were homogenized in 5 mL of lysis buffer (50 Mm Tris, 150 mM NaCl adjusted to pH 7.4) and centrifuged at 8000 g for 10 min.[18],[21] The supernatant was collected for the determination of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione reductase (GSH) levels.

Biochemical assays

Estimation of creatinine level

The concentration of Cr in plasma was measured by the Jaffe method using commercial diagnostic kits (Ref. 304331) purchased from Biomagreb (Ariana, Tunisia).

The MDA level in renal tissues was determined spectrophotometrically according to Draper and Hadley.[23]

Anti-oxidant markers in the renal tissues

The SOD activity was estimated according to Beauchamp and Fridovich[24] and GSH activity was assayed by the method of Ellman[25] modified by Jollow et al.[26]

Histopathological examination

For light microscopic examination, kidneys removed from the control and tested rats were cleaned and fixed in 10% buffered formalin solution. They were then embedded in paraffin and stained with hematoxylin-eosin for histopathological studies. All sections were evaluated for the degree of tubular and glomerular injury and necrosis.

Statistical Analysis

Data are expressed as mean ± standard deviation. The statistical significance between experimental groups was assessed by one-way analysis of variance followed by Tukey post hoc test. Statistical significance was set at P <0.05.

Results

Plasma creatinine level

The pCr level did not show any significant change between the different experimental groups [Table 1].

Table 1: Variation of plasma creatinine and urinary N-acetyl-b-D-glucosaminidase levels in different experimental groups of rats.

Urinary NAG levels increased significantly by 29% and 43%, respectively, in groups receiving 300,000 IU/kg/day and 450,000 IU/ kg/day of CMS, compared to controls. In animals with spontaneous renal recovery, the NAG decreased but without significant change. However, treatment with vit E significantly reduced the level of NAG [Table 1].

Lipid peroxidation in kidney

The renal tissue MDA levels increased significantly by 34% and 56%, respectively, after administration of 300,000 and 450,000 IU/kg/ day of CMS, compared to controls. Two weeks after stopping CMS, the MDA levels showed a slight decrease. However, treatment with vit E significantly reduced lipid peroxidation by 22% and 21%, respectively, compared to the CMS groups [Figure 1].

The activities of SOD and GSH in renal tissues declined after administration of CMS, the highest being in the 450,000 IU/kg/day group, compared to the controls. Two weeks after stopping CMS, the SOD and GSH activities showed a slight increase. However, treatment with vit E restored these activities, compared to the CMS group [Figure 2]a and [Figure 2]b.

The administration of 300,000 IU/kg/day of CMS for seven days led to a slight focal tubular dilatation. The severity of renal damage appeared to be more prominent in kidneys of the 450,000 IU/kg/day group, with acute tubular necrosis. In addition, we observed a significant increase of urine NAG, while the pCr remained normal. Urine NAG appears, therefore, to be more sensitive than pCr for early detection of proximal tubular damage due to colistin, as mentioned previously.[9],[27] The primary toxic effect of aminoglycosides is on the lysosomal system within the proximal tubule. NAG is a lysosomal enzyme present in high concentrations in the proximal tubular cells. The elevated urinary level of this enzyme reflects, therefore, renal tubular injury. Furthermore, we observed a significant increase in MDA level and a decline of SOD and GSH activities in the renal tissue of groups exposed to colistin. Thus, these findings support the role of oxidative stress on nephrotoxic effects due to colistin; oxidative stress has been reported in nephrotoxicity induced by numerous drugs.[28],[29],[30] Indeed, the proximal tubular cell lesions, evidenced by the rise of urinary NAG, could be the consequence of the excessive production of free radicals and the exhaustion of anti-oxidant enzymes.

After two weeks of stopping colistin, spontaneous evolution of tubular damage showed no significant histological improvement. However, urinary NAG and oxidative stress markers revealed a tendency to decrease. An eventual restitution of renal tissue would be therefore possible within more than two weeks. KochWeser et al[8] reported that renal dysfunction could progress for about two weeks after stopping colistin and usually resolves in three to nine weeks. In the clinical setting, based on the normalization of pCr, the authors estimated that renal reversibility following cessation of colistin might be resolved in one month or five to six weeks.[7],[8] However, pCr is not sensitive enough for better estimation of renal dysfunction; Ghlissi et al,[17] Yousef et al[10] and Wallace et al[6] demonstrated that the administration of high doses of colistin in rats led to severe tubular damage but without change in pCr. Interestingly, the reversibility of interstitial renal damage described by Kallel et al[31] concerns the immunoallergic mechanism damage and not the direct toxicity as elaborated here.

If we supported the involvement of oxidative stress in nephrotoxic effect of colistin, it would be conceivable to find an improvement in tubular damage after antioxidant treatment. Indeed, treatment for two weeks with vit E, following discontinuation of colistin, revealed significant renal regeneration. Histological and biochemical recovery seem to be total in the 300,000 IU/kg/day group. Renal amelioration might therefore be due to the antioxidant effect of vit E in neutralizing free radicals generated by colistin. Vit E is a fat-soluble antioxidant that can scavenge free radicals and inhibit the propagation of membrane lipid peroxidation.[32],[33] The decline of MDA level and the rise of SOD and GSH activities in renal tissue explain the curative effect of vit E. Additionally, the decreased level of NAG indicates the regeneration of tubular cell damage. Thus, treatment with vit E accelerated at least in part the reversibility of renal lesions compared to spontaneous evolution.

In conclusion, after two weeks of stopping colistin, spontaneous renal reversibility shows no significant amelioration. Treatment with vit E improves renal recovery. The curative effect of vit E might be related to the reduction of free radicals.

Acknowledgment

The authors are grateful to Professor Bou Yahia Moufida for assistance in writing this article.